Prevention of Chlamydia trachomatis (CT) infection is a public health priority because it causes reproductive sequelae. Since CT control programs have not reduced infection rates, a CT vaccine is needed. Our long-term goal is to generate knowledge that will advance CT vaccine development. One obstacle to CT vaccine development had been identifying immune and clinical correlates of protection in humans. We showed that CT- specific systemic CD4+ IFN-g is a correlate of immune protection in women and that spontaneous clearance of CT infection before treatment and absence of reinfection after treatment are clinical correlates of protection. With sparse data on CT-specific mucosal cellular immune responses in women, we developed a novel approach for studying these responses using mucosal mononuclear cells (MMCs) from menstrual blood (MB). Another obstacle in CT vaccine development has been identifying CT antigens that induce protective immunity. Using an immunoproteomic approach, Dr. Brunham?s laboratory identified five CT outer membrane proteins (OMPs) that elicited strong T helper type 1 (Th1) responses. They tested a CT and C. muridarum subunit vaccine that combined these OMPs with a Th1 polarizing adjuvant and found it accelerated infection clearance in mice. The potential immunogenicity of these CT OMPs and need for a CT vaccine provides strong rationale for extending immune studies of these promising CT antigens and others to humans. The major objective of our proposal (in response to FOA PA-19-096) is to study cellular and antibody responses to promising CT vaccine candidate antigens. Our hypothesis is that Th1 cytokine responses against candidate CT antigens will be associated with clinical correlates of protection and may correlate with functional antibody responses against CT organisms (EBs). Our study has 3 aims: Aim 1 - Investigate systemic cellular and humoral immune responses to candidate CT vaccine antigens and their association with clinical correlates of protection against CT in women. Peripheral blood mononuclear cells (PBMCs) from women with spontaneously cleared vs. persisting CT infection and women without vs. with CT reinfection will be stimulated ex vivo with promising CT vaccine candidate antigens and CD4+ and CD8+ T cell responses measured by intracellular cytokine staining (ICS). Sera will be tested for IgG to CT antigens and functional antibody responses to CT EBs. Aim 2 - Evaluate mucosal T cell and antibody responses induced by candidate CT vaccine antigens and their association with clinical correlates of protection against CT in women. From Aim 1 participants, MMCs from MB will be stimulated ex vivo with CT antigens and CD4+ and CD8+ T cell responses measured by ICS. Mucosal antibodies to CT antigens will be tested. Aim 3 - Use an in vitro human dendritic cell / CD4+ T cell co-culture method (MIMIC system) to confirm antigenicity and immunogenicity of promising candidate CT vaccine antigens in women. PBMCs from CT nave women will be used in the MIMIC system to evaluate antigenicity and immunogenicity (when given with adjuvant) of the candidate CT antigens. Study findings may lead to a CT vaccine formulation for a phase I study.